Bacillus thuringiensis isolates active against weevils

ABSTRACT

The subject invention concerns the discovery of  Bacillus thuringiensis  isolates with advantageous activity against weevils. In preferred embodiments of the invention,  B.t.  isolates, or toxins therefrom, are used to control alfalfa weevils, boll weevils, and/or rice water weevils. The toxins can be administered to the pests through a variety of methods including the transformation of bacteria or plants to produce the weevil-active toxins.

CROSS-REFERENCE TO A RELATED APPLICATION

This is a divisional of Ser. No. 09/737,228, filed Dec. 14, 2000 nowU.S. Pat. No. 6,605,462, which was a divisional of Ser. No. 09/401,890,filed Sep. 23, 1999, now U.S. Pat. No. 6,180,775, which was acontinuation of Ser. No. 09/005,280, filed Jan. 9, 1998, now abandoned,which was a continuation of Ser. No. 08/399,311, filed Mar. 6, 1995, nowU.S. Pat. No. 5,707,619.

FIELD OF THE INVENTION

The present invention relates to methods of controlling weevils. Inparticular, δ-endotoxins of Bacillus thuringiensis (B.t.) have beendiscovered to control rice water weevils, alfalfa weevils, and bollweevils.

BACKGROUND OF THE INVENTION

The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive,spore-forming bacterium characterized by parasporal crystalline proteininclusions. These inclusions often appear microscopically asdistinctively shaped crystals. The proteins can be highly toxic to pestsand specific in their toxic activity. Certain B.t. toxin genes have beenisolated and sequenced, and recombinant DNA-based B.t. products havebeen produced and approved for use. In addition, with the use of geneticengineering techniques, new approaches for delivering B.t. endotoxins toagricultural environments are under development, including the use ofplants genetically engineered with endotoxin genes for insect resistanceand the use of stabilized intact microbial cells as B.t. endotoxindelivery vehicles (Gaertner and Kim, 1988). Thus, isolated B.t.endotoxin genes are becoming commercially valuable.

Until the last fifteen years, commercial use of B.t. pesticides has beenlargely restricted to a narrow range of lepidopteran (caterpillar)pests. Preparations of the spores and crystals of B. thuringiensis var.kurstaki have been used for many years as commercial insecticides forlepidopteran pests. For example, B. thuringiensis var. kurstaki HD-1produces a crystal called a δ-endotoxin which is toxic to the larvae ofa number of lepidopteran insects.

In recent years, however, investigators have discovered B.t. pesticideswith specificities for a much broader range of pests. For example, otherspecies of B.t., namely B.t. var. israelensis and B.t. var. tenebrionis(a.k.a. M-7, a.k.a. B.t. var. san diego), have been used commercially tocontrol insects of the orders Diptera and Coleoptera, respectively(Gaertner, 1989). See also Couch, 1980 and Beegle, 1978. Krieg et al.,1983, describe Bacillus thuringiensis var. tenebrionis, which isreportedly active against two beetles in the order Coleoptera. These arethe Colorado potato beetle, Leptinotarsa decemlineata, and the beetleAgelastica alni.

Recently, new subspecies of B.t. have been identified, and genesresponsible for active δ-endotoxin proteins have been isolated (Höfteand Whiteley, 1989). Höfte and Whiteley classified B.t. crystal proteingenes into 4 major classes. The classes were CryI(Lepidoptera-specific), CryII (Lepidoptera- and Diptera-specific),CryIII (Coleoptera-specific), and CryIV (Diptera-specific). Prefontaineet al., 1987, describe probes useful in classifying lepidopteran-activegenes. The discovery of strains specifically toxic to other pests hasbeen reported (Feitelson et al., 1992).

B.t. crystalline toxins are generally recognized as being protoxins,requiring either particular physicochemical conditions (i.e., pH, redox,ionic strength), or the action of certain proteases, or both, togenerate an active toxin (Höfte and Whiteley, 1989). In most cases, theinsect supplies conditions for activation of the toxin; however, caseshave been documented where pre-solubilization or pre-proteolysis havebeen necessary for optimum activity (Jacquet et al., 1987) or detectionof activity (Höfte et al., 1992).

The cloning and expression of a B.t. crystal protein gene in Escherichiacoli has been described in-the published literature (Schnepf andWhiteley, 1981). U.S. Pat. Nos. 4,448,885 and 4,467,036 both disclosethe expression of B.t. crystal proteins in E. coli. U.S. Pat. Nos.4,797,276 and 4,853,331 disclose B. thuringiensis var. tenebrionis(a.k.a. B.t. san diego, a.k.a. M-7) which can be used to controlcoleopteran pests in various environments. U.S. Pat. No. 4,918,006discloses Bacillus thuringiensis var. israelensis toxins which areactive against dipteran pests and reports that a protein of about 27 kD,and fragments thereof, are responsible for the dipteran activity. U.S.Pat. No. 4,849,217 discloses B.t. isolates which have activity againstthe alfalfa weevil. U.S. Pat. No. 5,151,363 and U.S. Pat. No. 4,948,734disclose certain isolates of B.t. which have activity against nematodes.As a result of extensive research and investment of resources, otherpatents have issued for new B.t. isolates and new uses of B.t. isolates.However, the discovery of new B.t. isolates and new uses of known B.t.isolates remains an empirical, unpredictable art.

The alfalfa weevil, Hypera postica, and the closely related Egyptianalfalfa weevil, Hypera brunneipennis, are the most important insectpests of alfalfa grown in the United States, with 2.9 million acresinfested in 1984. An annual sum of 20 million dollars is spent tocontrol these pests. The Egyptian alfalfa weevil is the predominantspecies in the southwestern U.S., where it undergoes aestivation (i.e.,hibernation) during the hot summer months. In all other respects, it isidentical to the alfalfa weevil, which predominates throughout the restof the U.S.

The larval stage is the most damaging in the weevil life cycle. Byfeeding at the alfalfa plant's growing tips, the larvae causeskeletonization of leaves, stunting, reduced plant growth, and,ultimately, reductions in yield. Severe infestations can ruin an entirecutting of hay. The adults, also foliar feeders, cause additional, butless significant, damage.

The rice water weevil, Lissorhoptrus aryzophilus, is a major insect pestof rice in North America and Southeast Asia. See Smith, M. C. (1983)“The Rice Water Weevil, Lissorhoptrus oryzophilus Kuschel,” Exotic PlantQuarantine Pests and Possibilities for Introduction of Plant Materials,pp. 3–9. The rice water weevil can be directly responsible for averageyield reductions of 10% or more if not treated with the properinsecticides. Rice water weevil larvae cause significant damage to theroot systems of cultivated rice. Adult rice water weevils are small,black, oblong weevils (2.8–3.2 mm long×1.2–1.8 mm wide) with grayscales. Adults feed on rice by rasping away the leaf epidermis leavingskeletonized longitudinal slits on the upper leaf blades. Adult weevilsappear to prefer two-week old rice plants over those of seven-week oldplants and increased levels of nitrogen fertilizers increase the levelof feeding. Adults also feed on individual grains of headed riceconsuming the floral part or the endosperm of the developing ricekernel. Weevils enter a true diapause, fly to hibernation sites as earlyas July and overwinter in bunch grasses, Spanish moss and ground trash.Upon emerging in the spring the weevils migrate to flooded rice fieldswhere they mate. Eggs are deposited in submerged leaf sheaths on thelower part of the rice plant and hatch within 4–9 days.

The rice water weevil larvae have 4 instars. The length of each instarphase is temperature dependent and under normal field conditions thelarval stages last about 27 days. The larvae have paired dorsal trachealhooks which function as modified spiracles. The apical segment of thehook is heavily sclerotized and is used to pierce root tissue andsequester air. This allows the larvae to live below the water surface.The pupae form in an oval, water-tight mud cell and resembles the adultin size and shape but is white in color. The duration of the pupal stageis seven days at 27° C. Two and three generations per season have beenreported.

Control of the rice water weevil is difficult due to its terrestrial,aquatic and soil habitats. Chemical insecticides have been used in thepast with limited success. Native resistant rice cultivars are beingsought with only low to moderate resistant lines being discovered.

The International Rice Research Newsletter (1983) Vol. 8, No. 6, pp.16–17, reports pathogens and nematodes for the control of rice waterweevil. Various strains of fungi (Beauveria bassiana and Metarrhiziumanisopliae) and nematodes were shown to control rice water weevils. Noneof the B.t. biopreparations controlled rice water weevils when appliedto 35 day old rice plants as a foliar spray at 0.6, 1.2 and 2.4 kg/ha.

It has been unexpectedly discovered that B.t. δ-endotoxins are effectivein controlling rice water weevils contrary to published reports thatB.t. biopreparations were ineffective in controlling this pest.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns the use of Bacillus thuringiensis (B.t.)isolates and δ-endotoxins therefrom to control weevil pests.Specifically exemplified herein is the use of B.t. isolates and toxinsto control alfalfa weevils, boll weevils, and rice water weevils.

In one preferred embodiment of the present invention, B.t. δ-endotoxinsare employed to control rice water weevils. The B.t. δ-endotoxin isadministered to rice water weevils in a manner wherein the rice waterweevils ingest the toxin. The B.t. δ-endotoxins may be applied as afoliar spray to rice crops where adult weevils graze. Also, a B.t.structural gene may be inserted into the rice plant genome to producethe δ-endotoxin in planta throughout the plant or in specific planttissues to control both adults and larvae.

In one preferred embodiment of the subject invention, B.t. toxins of theCryIII class are used to control weevil pests. For example, a CryIIIB.t. isolate HD511 has shown excellent activity against the rice waterweevil.

Further exemplified herein is the use of B.t. isolates PS50C, PS201T6,KB92, PS86B1, PS101Z2, PS50B, PS204G4, PS204G6, PS167P, PS192N1,PS201L1, PS169E, PS177G, PS196S1, and PS73E to control weevils.

In another embodiment of the subject invention, the materials andmethods set forth herein pertain particularly to Bacillus thuringiensisvar. israelensis, or toxins therefrom, to control weevils. A relatedembodiment concerns the use of an activated toxin from B.t. isolatePS201T6 to control weevils.

As described herein, the toxins from the B.t. strains HD511, PS73E,PS192N1 and PS201T6 (activated toxin) are particularly effective incontrolling rice water weevils. The PS201T6 activated toxin is alsohighly active against cotton boll weevil.

The subject invention also includes the use of variants of theexemplified B.t. isolates which have substantially the same pesticidalproperties as the exemplified isolate. These variants include mutants.Procedures for making mutants are well known in the microbiological art.Ultraviolet light and chemical mutagens such as nitrosoguanidine areused extensively toward this end.

Recombinant hosts which have been transformed to express B.t. toxins canalso be used according to the subject invention. These recombinant hostsmaybe, for example, microorganisms or plants. Specifically exemplifiedherein is the recombinant microorganism designated MR506.

According to the subject invention, weevils may be controlled using theisolates themselves, variants of the isolates, δ-endotoxins obtainedfrom said isolates, commercial preparations made from cultures of theseisolates, or toxins produced by DNA of these isolates. In oneembodiment, the toxins may be produced by DNA which has been transformedinto another host. Further, the invention also includes the treatment ofsubstantially intact cells of either a B.t. isolate or recombinant cellscontaining DNA from a B.t. isolate, to prolong the pesticidal activitywhen the substantially intact cells are applied to the environment of atarget pest.

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns the discovery of a new method forcontrolling weevils which involves contacting the weevils with aBacillus thuringiensis (B.t.) isolate or a δ-endotoxin therefrom.Several B.t. isolates with excellent activity against weevils aredescribed herein.

In one specific embodiment of the present invention, one or more B.t.δ-endotoxins are administered to rice water weevils to control this pestin rice crops. The B.t. δ-endotoxin is administered in a manner whereinthe weevils ingest the toxin to allow the toxin to disrupt theepithelial cell wall of the midgut. As would be appreciated by oneskilled in the art, the exact method of administration is not critical.For example, the B.t. δ-endotoxins can be administered as a foliar sprayonto rice crops. This method of administration is effective incontrolling adult weevils that feed on the leaves of rice plants.Another method of administration is accomplished by inserting a B.t.insecticidal structural gene into a rice plant genome whereby theδ-endotoxin is produced in vivo in the plant. Both adults and larvaegrazing on the transgenic plant will ingest the δ-endotoxin.Advantageously, tissue-specific promoters can be employed to drive theexpression of the B.t. gene so that the toxin is present in the tissuewhich is most likely to be eaten by the weevil. For example, rootspecific promoters can be employed to provide control of larvae and leafspecific promoters can be employed to control adult weevils.

In one embodiment of the subject invention, the B.t. isolates utilizedare B.t. var. israelensis, or toxins therefrom. The israelensis varietyis well known and readily recognized by those skilled in this art.Characteristics generally associated with the israelensis category ofB.t. include dipteran activity, H14 serotype, and a protein patternwhich includes an approximately 28 to 33 kD protein and, generally,additional proteins of about 70 kD and 130 kD. Other B.t. varietieswhich express israelensis-type toxins can also be used according to thesubject invention. Such toxins would have a size similar to the toxinsproduced by B.t.i. and a similar activity profile. B.t. isolates of thevar. morrisoni serotype 8a,8b have, for example, been reported toexpress B.t.i.-typetoxins. An example of such an isolate is PS71M3. Asused herein, the term “Bacillus thuringiensis var. israelensis toxin”includes toxins which are similar or related to toxins expressed byB.t.i. but which happen to be expressed by a different variety of B.t.

In one preferred embodiment of the subject invention, an activated toxinfrom B.t. isolate PS201T6 is used to achieve excellent control ofweevils. Similarly, toxins from B.t.i. may also be activated by, forexample, growing a Bacillus thuringiensis var. israelensis underconditions which facilitate the activation of said toxin by the actionof compounds which exist naturally or are produced in said culture.Activation may also be achieved by adding a compound to a Bacillusthuringiensis var. israelensis culture, or a supernatant thereof,wherein said compound participates in the activation of said toxineither through direct action on said toxin or by facilitating the actionof a second compound. The additional compound may be, for example, aprotease, or a compound which raises the pH of the culture orsupernatant.

The cultures disclosed in this application have been deposited in theAgricultural Research Service Patent Culture Collection (NRRL), NorthernRegional Research Center, 1815 North University Street, Peoria, Ill.61604, USA.

TABLE 1 Culture deposit information Isolate Deposit No. Deposit Date B.t. japonensis bui bui FERM BP-3465 Jun. 26, 1992 (KB92) HD511 HowardDulmage Collection, Texas PS33F2 NRRL B-18244 Jul. 28, 1987 PS50B NRRLB-21656 Feb. 19, 1997 PS50C NRRL B-18746 Jan. 9, 1991 PS73E NRRL B-21417Mar. 8, 1995 PS86B1 NRRL B-18299 Feb. 2, 1988 PS101Z2 NRRL B-18890 Oct.1, 1991 PS167P NRRL B-18681 Jul. 17, 1990 PS169E NRRL B-18682 Jul. 17,1990 PS177G NRRL B-18684 Jul. 17, 1990 PS192N1 NRRL B-18721 Oct. 5, 1990PS196S1 NRRL B-18748 Jan. 9, 1991 PS201L1 NRRL B-18749 Jan. 9, 1991PS201T6 NRRL B-18750 Jan. 9, 1991 PS204G4 NRRL B-18685 Jul. 17, 1990PS204G6 NRRL B-18686 Jul. 17, 1990

The subject cultures have been deposited under conditions that assurethat access to the cultures will be available during the pendency ofthis patent application to one determined by the Commissioner of Patentsand Trademarks to be entitled thereto under 37 CFR 1.14 and 35 U.S.C.122. The deposits are available as required by foreign patent laws incountries wherein counterparts of the subject application, or itsprogeny, are filed. However, it should be understood that theavailability of the deposits does not constitute a license to practicethe subject invention in derogation of patent rights granted bygovernmental action.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of a deposit, and in any case, for a period of at least thirty(30) years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposit(s) should the depository beunable to furnish a sample when requested, due to the condition of thedeposit(s). All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

Certain B.t. isolates, genes, and toxins useful according to the subjectinvention have been previously described in issued U.S. patents orpublished international patent documents. These isolates and theirpreviously-discovered uses are tabulated below:

TABLE 2 Isolate Patent or Publication Activity KB92 (bui bui) 5,359,048coleopterans MR506 WO93/04587 lepidopterans HD511 5,262,324 coleopterans5,286,486 coleopterans PS50C 5,185,148 scarabs 5,262,158 mites 5,277,905coleopterans 5,286,485 lepidopterans PS86B1 4,966,765 coleopterans5,100,665 lesser mealworms 5,185,148 scarabs PS167P 5,151,363 nematodesPS169E 5,151,363 nematodes PS177G 5,151,363 nematodes PS192N1 5,262,160dipterans 5,273,746 lice PS196S1 5,298,245 dipterans PS201L1 5,298,245dipterans PS201T6 5,273,746 lice 5,298,245 dipterans 5,302,387cockroaches PS204G4 5,151,363 nematodes PS204G6 5,151,363 nematodes5,273,746 lice PS33F2 5,151,363 nematodes 4,849,217 coleopterans

Nothing in those documents describes or suggests the unique activitydescribed herein. The disclosures of those patents are incorporatedherein by reference. Those patents contain, for example, descriptions oftoxins and genes from the indicated isolates. Those toxins and genes canbe used by one skilled in the art in conjunction with the teachingsprovided herein to control weevil pests.

Genes and toxins. In one embodiment of the subject invention, geneswhich encode B.t. toxins active against weevil pests are used totransform a suitable host. The genes and toxins useful according to thesubject invention include not only the full length sequences but alsofragments of these sequences, variants, mutants, and fusion proteinswhich retain the characteristic pesticidal activity of the toxinsspecifically exemplified. As used herein, the terms “variants” or“variations” of genes refer to nucleotide sequences which encode thesame toxins or which encode equivalent toxins having pesticidalactivity. As used herein, the term “equivalent toxins” refers to toxinshaving the same or essentially the same biological activity against thetarget pests as the claimed toxins.

It should be apparent to a person skilled in this art that genesencoding weevil-active toxins can be identified and obtained throughseveral means. The genes may be obtained from the isolates deposited ata culture depository as described above. These genes, or portions orvariants thereof, may also be constructed synthetically, for example, byuse of a gene synthesizer. Variations of genes may be readilyconstructed using standard techniques for making point mutations. Also,fragments of these genes can be made using commercially availableexonucleases or endonucleases according to standard procedures. Forexample, enzymes such as Bal31 or site-directed mutagenesis can be usedto systematically cut off nucleotides from the ends of these genes.Also, genes which encode active fragments may be obtained using avariety of restriction enzymes. Proteases may be used to directly obtainactive fragments of these toxins.

Equivalent toxins and/or genes encoding these equivalent toxins can bederived from B.t. isolates and/or DNA libraries using the teachingsprovided herein. There are a number of methods for obtaining thepesticidal toxins of the instant invention. For example, antibodies tothe pesticidal toxins disclosed and claimed herein can be used toidentify and isolate other toxins from a mixture of proteins.Specifically, antibodies may be raised to the portions of the toxinswhich are most constant and most distinct from other B.t. toxins. Theseantibodies can then be used to specifically identify equivalent toxinswith the characteristic activity by immunoprecipitation, enzyme linkedimmunosorbent assay (ELISA), or Western blotting. Antibodies to thetoxins disclosed herein, or to equivalent toxins, or fragments of thesetoxins, can readily be prepared using standard procedures in this art.The genes which encode these toxins can then be obtained from themicroorganism.

Fragments and equivalents which retain the pesticidal activity of theexemplified toxins would be within the scope of the subject invention.Also, because of the redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate these alternative DNA sequences encoding the same, or essentiallythe same, toxins. These variant DNA sequences are within the scope ofthe subject invention. As used herein, reference to “essentially thesame” sequence refers to sequences which have amino acid substitutions,deletions, additions, or insertions which do not materially affectpesticidal activity.

A further method for identifying the toxins and genes of the subjectinvention is through the use of oligonucleotide probes. These probes arenucleotide sequences having a means for detection. As is well known inthe art, if the probe molecule and nucleic acid sample hybridize byforming a strong bond between the two molecules, it can be reasonablyassumed that the probe and sample have substantial homology. The probe'smeans of detection provides a means for determining in a known mannerwhether hybridization has occurred. Such a probe analysis provides arapid method for identifying toxin-encoding genes of the subjectinvention. The nucleotide segments which are used as probes according tothe invention can be synthesized by use of DNA synthesizers usingstandard procedures. These nucleotide sequences can also be used as PCRprimers to amplify genes of the subject invention.

Recombinant hosts. The toxin-encoding genes harbored by the isolatesdisclosed herein can be introduced into a wide variety of microbial orplant hosts. Expression of the toxin gene results, directly orindirectly, in the production of the toxin. With suitable microbialhosts, e.g., Pseudomonas, the microbes can be applied to the situs ofthe pest, where they will proliferate and be ingested by the pest,resulting in control of the pest. Alternatively, the microbe hosting thetoxin gene can be treated under conditions that prolong the activity ofthe toxin and stabilize the cell. The treated cell, which retains thetoxic activity, then can be applied to the environment of the targetpest.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is advantageous to use certain host microbes. For example,microorganism hosts can be selected which are known to occupy the pest'shabitat. Microorganism hosts may also live symbiotically with a specificspecies of weevil. These microorganisms are selected so as to be capableof successfully competing in the particular environment with thewild-type microorganisms, provide for stable maintenance and expressionof the gene expressing the polypeptide pesticide, and, desirably,provide for improved protection of the pesticide from environmentaldegradation and inactivation.

A wide variety of ways are available for introducing a B.t. geneencoding a toxin into a microorganism host under conditions which allowfor stable maintenance and expression of the gene. These methods arewell known to those skilled in the art and are described, for example,in U.S. Pat. No. 5,135,867, which is incorporated herein by reference.

Furthermore, materials and methods for introducing B.t. genes intoplants in order to confer upon such plants the ability to produceinsecticidal toxins is well known in the art. In a preferred embodiment,the B.t. genes are modified to facilitate optimal stability andexpression in the selected plant host. In this regard, U.S. Pat. No.5,380,831 is incorporated herein by reference.

Treatment of cells. As mentioned above, B.t. or recombinant cellsexpressing a B.t. toxin can be treated to prolong the toxin activity andstabilize the cell by forming a cellular microcapsule. The pesticidemicrocapsule that is formed comprises the B.t. toxin within a cellularstructure that has been stabilized and will protect the toxin when themicrocapsule is applied to the environment of the target pest. Suitablehost cells may include either prokaryotes or eukaryotes, normally beinglimited to those cells which do not produce substances toxic to higherorganisms, such as mammals. However, organisms which produce substancestoxic to higher organisms could be used, where the toxic substances areunstable or the level of application sufficiently low as to avoid anypossibility of toxicity to a mammalian host. As hosts, of particularinterest will be the prokaryotes and the lower eukaryotes, such asfungi.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

Treatment of the microbial cell, e.g., a microbe containing the B.t.toxin gene, can be by chemical or physical means, or by a combination ofchemical and/or physical means, so long as the technique does notdeleteriously affect the properties of the toxin, nor diminish thecellular capability of protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17–80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as glutaraldehyde;anti-infectives, such as zephiran chloride and cetylpyridinium chloride;alcohols, such as isopropyl and ethanol; various histologic fixatives,such as Lugol iodine, Bouin's fixative, various acids, and Helly'sfixative (See: Humason, 1967); or a combination of physical (heat) andchemical agents that preserve and prolong the activity of the toxinproduced in the cell when the cell is administered to the host animal.Examples of physical means are short wavelength radiation such asgamma-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like. Methods for treatment of microbial cellsare disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which areincorporated herein by reference.

Growth of cells. The cellular host containing the B.t. insecticidal genemay be grown in any convenient nutrient medium, where the DNA constructprovides a selective advantage, providing for a selective medium so thatsubstantially all or all of the cells retain the B.t. gene. These cellsmay then be harvested in accordance with conventional ways.Alternatively, the cells can be treated prior to harvesting.

The B.t. cells of the invention can be cultured using standard art mediaand fermentation techniques. Upon completion of the fermentation cyclethe bacteria can be harvested by first separating the B.t. spores andcrystals from the fermentation broth by means well known in the art. Therecovered B.t. spores and crystals can be formulated into a wettablepowder, liquid concentrate, granules or other formulations by theaddition of surfactants, dispersants, inert carriers, and othercomponents to facilitate handling and application for particular targetpests. These formulations and application procedures are all well knownin the art.

Formulations. Formulated bait granules containing an attractant andspores and crystals of the B.t. isolates, or recombinant microbescomprising the genes obtainable from the B.t. isolates disclosed herein,can be applied to the environment of the weevil. The bait may be appliedliberally since the toxin does not affect animals or humans. Product mayalso be formulated as a spray or powder. The B.t. isolate or recombinanthost expressing the B.t. gene may also be incorporated into a bait orfood source for the weevil.

As would be appreciated by a person skilled in the art, the pesticidalconcentration will vary widely depending upon the nature of theparticular formulation, particularly whether it is a concentrate or tobe used directly. The pesticide will be present in at least 1% by weightand may be 100% by weight. The dry formulations will have from about1–95% by weight of the pesticide while the liquid formulations willgenerally be from about 1–60% by weight of the solids in the liquidphase. Formulations that contain cells will generally have from about10² to about 10⁴ cells/mg. These formulations will be administered atabout 50 mg (liquid or dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the weevils, e.g.on plant foliage.

Mutants. Mutants of the isolates described herein can be made byprocedures well known in the art. For example, an asporogenous mutantcan be obtained through ethylmethane sulfonate (EMS) mutagenesis of anovel isolate. The mutants can be made using ultraviolet light andnitrosoguanidine by procedures well known in the art.

A smaller percentage of the asporogenous mutants will remain intact andnot lyse for extended fermentation periods; these strains are designatedlysis minus (−). Lysis minus strains can be identified by screeningasporogenous mutants in shake flask media and selecting those mutantsthat are still intact and contain toxin crystals at the end of thefermentation. Lysis minus strains are suitable for a cell treatmentprocess that will yield a protected, encapsulated toxin protein.

To prepare a phage resistant variant of said asporogenous mutant, analiquot of the phage lysate is spread onto nutrient agar and allowed todry. An aliquot of the phage sensitive bacterial strain is then plateddirectly over the dried lysate and allowed to dry. The plates areincubated at 30° C. The plates are incubated for 2 days and, at thattime, numerous colonies could be seen growing on the agar. Some of thesecolonies are picked and subcultured onto nutrient agar plates. Theseapparent resistant cultures are tested for resistance by cross streakingwith the phage lysate. A line of the phage lysate is streaked on theplate and allowed to dry. The presumptive resistant cultures are thenstreaked across the phage line. Resistant bacterial cultures show nolysis anywhere in the streak across the phage line after overnightincubation at 30° C. The resistance to phage is then reconfirmed byplating a lawn of the resistant culture onto a nutrient agar plate. Thesensitive strain is also plated in the same manner to serve as thepositive control. After drying, a drop of the phage lysate is plated inthe center of the plate and allowed to dry. Resistant cultures showed nolysis in the area where the phage lysate has been placed afterincubation at 30° C. for 24 hours.

Following are examples which illustrate procedures, including the bestmode, for practicing the invention. These examples should not beconstrued as limiting. All percentages are by weight and all solventmixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Culturing B.t. Isolates

A subculture of the B.t. isolate can be used to inoculate the followingpeptone, glucose, and salts medium.

Bacto Peptone 7.5 g/l Glucose 1.0 g/l KH₂PO₄ 3.4 g/l K₂HPO₄ 4.35 g/lSalt Solution 5.0 ml/l CaCl₂ Solution 5.0 ml/l Salts Solution (100 ml)MgSO₄ .7H₂O 2.46 g MnSO₄ .H₂O 0.04 g ZnSO₄ .7H₂O 0.28 g FeSO₄ .7H₂O 0.40g CaCl₂ Solution (100 ml) CaCl₂ .2H₂O 3.66 g pH 7.2

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The B.t. spores and crystals, obtained in the above fermentation, can beisolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Production of Activated 201T6 Toxin (201T6-D)

Activated 201T6 toxin can be produced by a variety of methods whichresult in truncation of the 201T6 toxin. In this regard, reference canbe made to WO95/02693. In one such method, cultures of PS201T6 wereharvested by centrifugation and resuspended to 1/9th to 1/25th of theiroriginal culture volume in 0.1 Na₂CO₃/NaHCO₃ pH 11.0 containing 0.5mg/ml pronase E (Sigma Chemical Company, P-5147 Type XIV bacterialprotease from Streptomyces griseus). The suspension was incubated at 37°C. overnight with mixing. The suspensions were dialyzed against 2changes of 50 to 100 volumes each of either distilled water or 0.1 MNa₂CO₃/NaHCO₃ pH 9.5 to yield “dialyzed suspensions.”

The suspension resulting from 0.1 M Na₂CO₃/NaHCO₃ pH 9.5 dialysis wascentrifuged to remove cells, spores, and debris. Additional purificationfrom spores and debris can be accomplished by filtration through aWhatman glass microfibre filter, a 0.8 micron cellulose acetate filter,and a 0.2 micron cellulose acetate filter to yield a “filteredsupernatant.”

Dried preparations of the processed toxin were prepared either before orafter filtration by dialyzing against 2 changes of 50 to 100 volumesdistilled water, followed by lyophilization (lyophilized,pronase-treated toxin).

EXAMPLE 3 Alternative Method for Production of Activated 201T6 Toxin

Cultures of PS201T6 were harvested by centrifugation and resuspended to1/9th to 1/25th of their original culture volume in 0.1 M Na₂CO₃/NaHCO₃,0.5% 2-mercaptoethanol, pH 11.0. The suspension was incubated for about2 hours at room temperature. The suspension was centrifuged to removecells, spores, and debris. Additional purification from spores anddebris can be accomplished by filtration through a Whatman glassmicrofibre filter, a 0.8 micron cellulose acetate filter, and a 0.2micron cellulose acetate filter to yield a “filtered supernatant.” Thesuspensions were dialyzed against 2 changes of 50 to 100 volumes each ofeither distilled water or 0.1 M Na₂CO₃/NaHCO₃ pH 9.5 to yield “dialyzedsuspensions.” Dried preparations of the processed toxin were preparedeither before or after filtration by dialyzing against two changes of 50to 100 volumes distilled water, followed by lyophilization. Materialprepared according to this procedure is referred to herein as 201T6-D.

EXAMPLE 4 Activity of B.t. Isolates Against Second Instar Alfalfa WeevilHypera postica

The preparations were tested against second instar alfalfa weevil by atop-load bioassay on artificial diet. The agar-based diet was dispensedinto individual wells of 96-well, flat-bottom plates, and allowed tosolidify before the application of the suspension to the surface of thediet. Trays were gently swirled on a rotary shaker to ensure dietsurface coverage with the suspensions. The toxin suspensions on thesurface of the diet were allowed to air-dry under a laminar flow hood. Asingle larva was infested into each well and the plate sealed by apolyester film with heat-sensitive adhesive. The assay trays were heldat 23° C., 70% RH, 8:16 day:night cycle for six days. The larvae wereobserved under a dissection microscope for mortality. Table 3 showsbioassay results from three screens using the artificial diet top-loadbioassay in 96-well trays. In Table 3, PS201T6 is the whole lyophilizedculture; PS201T6-D is prepared as described in Example 3.

TABLE 3 Bioassay results of B.t. strains against Hypera postica Screen 1Screen 2 Screen 3 Isolate μg/cm² % μg/cm² % μg/cm² % PS201T6 130 92 85100 5 100 65 75 32 100 2.4 93 18 100 1.2 50 16 100 0.6 18 8 100 0.3 25 4100 PS201T6-D 65 100 2 100 2 100 (activated) 32 100 1 78 1 100 16 1000.5 57 0.5 91 8 100 0.25 20 0.25 73 4 100 0.12 0 0.12 68 MR506 65 75 6570 65 66 32 25 32 46 32 31 16 33 16 25 16 100 8 8 8 37 8 85 4 8 4 13 442 KB92 130 27 130 50 130 87 65 10 65 0 65 75 HD-511 130 8 130 0 130 2565 8 65 14 65 6 Water 0 0 0 0 0 0

EXAMPLE 5 Activity of B.t. Isolates Against the Rice Water Weevil

B.t. isotates were evaluated for their insecticidal activity againstrice water weevil adults. The insects used in these experiments werecollected from rice at the Rice Research Station, Acadia Parish La. Theweevils were taken to a laboratory, placed in plastic storage boxeslined with moist paper towels and held at 27° C. with a 16 hourphotoperiod. Weevils were provided an excess of rice foliage (var.Cypress) which was replenished every two days. The rice from whichweevils were collected was not treated with insecticides during thegrowing season.

The plants used in the experiments were greenhouse grown Cypress rice. A16 hour photoperiod was provided by metal halide lamps. The temperaturein the greenhouse fluctuated from 27° C. at night to daytime highs inexcess of 32° C.

The B.t. formulations were made by adding 10 mg of B.t. isolate to a 2ml microcentrifuge tube containing 1 ml of a 0.1% aqueous solution ofBond adjuvant. The microcentrifuge tubes were sealed and sonicated for15 seconds, resulting in the formation of suspensions that were storedat 4° C. until used in the bioassays.

The bioassays were conducted by excising the newest unfurled leaves ofgreenhouse grown Cypress rice plants and wrapping them in moist papertowels. The excised leaves were taken to the laboratory and gently wipedwith a cotton ball saturated with a 0.1% solution of Bond adjuvantbefore being cut into 2.5–3.5 cm sections. The apical and basal 3 cm ofeach leaf was discarded. Individual leaf sections were placed intomicrocentrifuge tubes containing one of the B.t. isolate suspensions.The tubes were sealed and vortexed for 3–5 seconds to uniformly coat theleaf section with the isolate suspension. Leaf sections were placedindividually into 5 cm Petri dishes containing two filter paper disksmoistened with 8–10 drops of tap water. One adult rice water weevil wasadded to each dish and the dish was covered and sealed with “PARAFILM”brand film. The Petri dishes were maintained at 27° C. with a 16 hourphotoperiod. Every 24 hours the Petri dishes were opened and weevilmortality and extent of feeding were determined.

Percent mortality caused by the isolates is listed below. Alltreatments, with the exception of the negative controls caused somemortality to the weevils. The MR508 recombinant E. coli expresses atoxin obtainable from B.t. isolate PS33F2.

TABLE 4 Mortality % Treatment 2 day 4 day 1 Adjuvant  0 10 2 PS50C 20 403 MR506 30 50 4 201T6-D 80 100  5 HD511 60 80 7 KB92 20 40 7 PS86B1 3060 8 PS201T6 30 30 9 PS101Z2 10 60 10 PS50B 30 60 11 MR508 30 30 12PS204G4 20 30 13 PS204G6 30 50 14 PS167P 20 40 15 PS192NL 20 70 16PS201L1 30 30 17 PS169E 10 30 18 PS177G 30 40 19 PS196S1 30 30 20 PS73E40 70 21 untreated  0  0

EXAMPLE 6

Of particular interest according to the subject invention is the use ofCryIII B.t. toxins and cyt toxins to control weevils. As shown in Table5, toxins from several subclasses of CryIII toxins, as well as cyttoxins, can be used to effectively control weevils. The CryIII toxinsare well known to those skilled in the art. Of particular interest inthe subject invention are those toxins which have at least 50% aminoacid sequence identity with a toxin selected from the group consistingof CryIIIA, HD511, 50C, or KB92. Preferably, the sequence identity willbe more than 75%. The cyt toxins are also well known and are exemplifiedby, for example, cytA, cytB, and the 201T6 toxin. Again, greater then50% or 75% sequence identity with these toxins is preferred.

TABLE 5 Isolate B.t. toxin Percent Mortality (4 days) PS50C CryIII 40MR525 CryIIIA 40 PS86B1 CryIIIB 60 HD511 CryIII 80 PS50B CryIII 60PS201T6 cyt (201T6-D) 100 Untreated 0

EXAMPLE 7 Activity of Bacillus thuringiensis on the Cotton Boll Weevil

Strains of B.t. were tested for activity against boll weevil. The B.t.strains were provided as powders. Powders were weighed and suspended indistilled water, vortexed, and diluted in distilled water to the desiredconcentration.

Boll weevil eggs were received on diet from the USDA Boll Weevil RearingLaboratory, Stoneville, Miss., and maintained in a growth chamber at 30°C., with a 12:12 (L:D) photoperiod, until hatch. B.t. strains suspendedin 1 ml distilled water were incorporated into 50 ml modified VA+SB dietmaintained at 55° C. in 125 ml flasks. The diet/B.t. mixture was pouredinto 15×100 mm plastic petri dishes. A plastic grid was immediatelyinserted into each diet plate. Twenty-one second instar larvae wereplaced in each dish. Petri dishes containing diet, B.t., and larvae werecovered with parafilm and maintained in a growth chamber as describedabove. Mortality was evaluated at 24,48, and 72 hours. Plates werephotographed at 72 hours to obtain a qualitative estimate of the amountsof insect burrowing and feeding. Larvae were considered dead at 72 hoursif they did not respond to probes with a camel hair brush.

Particularly good activity was observed for B.t. isolate PS201T6 and thetoxin obtained from this isolate. In a preferred embodiment, the 201T6protein is processed using procedures such as those which are describedin Examples 2 and 3.

Activity against the cotton boll weevil was also observed for B.t.strain bui bui (KB92).

EXAMPLE 8 Insertion of Toxin Genes Into Plants

One aspect of the subject invention is the transformation of plants withgenes coding for a toxin active against weevils. The transformed plantsare resistant to attack by weevils.

Genes encoding weevil-active toxins, as disclosed herein, can beinserted into plant cells using a variety of techniques which are wellknown in the art. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, microinjection, bombardment, chemicalagent (PEG) assisted DNA uptake, or electroporation as well as otherpossible methods. If agrobacteria are used for the transformation, theDNA to be inserted has to be cloned into special plasmids, namely eitherinto an intermediate vector or into a binary vector. The intermediatevectors can be integrated into the Ti or Ri plasmid by homologousrecombination owing to sequences that are homologous to sequences in theT-DNA. The Ti or Ri plasmid also comprises the vir region necessary forthe transfer of the T-DNA. Intermediate vectors cannot replicatethemselves in agrobacteria. The intermediate vector can be transferredinto Agrobacterium tumefaciens by means of a helper plasmid(conjugation).

Binary vectors can replicate themselves both in E. coli and inagrobacteria. They comprise a selection marker gene and a linker orpolylinker which are framed by the right and left T-DNA border regions.They can be transformed directly into agrobacteria (Holsters et al.[1978] Mol. Gen. Genet. 163:181–187). The agrobacterium used as hostcell is to comprise a plasmid carrying a vir region. The vir region isnecessary for the transfer of the T-DNA into the plant cell.

The use of T-DNA for the transformation of plant cells has beenintensively researched and sufficiently described in EP 120 516; Hoekema(1 985) In: The Binary Plant Vector System, Offset-durkkerij KantersB.V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci.4:1–46; and An et al. (1985) EMBO J. 4:277–287.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there and, as a rule, does not come out again. Itnormally contains a selection marker that confers on the transformedplant cells resistance to a biocide or an antibiotic, such as kanamycin,G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. Theindividually employed marker should accordingly permit the selection oftransformed cells rather than cells that do not contain the insertedDNA.

The bacterium so transformed is used for the transformation of plantcells. Plant explants can advantageously be cultivated withAgrobacterium tumefaciens or Agrobacterium rhizogenes for the transferof the DNA into the plant cell. Whole plants can then be regeneratedfrom the infected plant material (for example, pieces of leaf, segmentsof stalk, roots, but also protoplasts or suspension-cultivatedcultivated cells) in a suitable medium, which may contain antibiotics orbiocides for selection. The plants so obtained can then be tested forthe presence of the inserted DNA. No special demands are made of theplasmids in the case of microinjection and electroporation. It ispossible to use ordinary plasmids, such as, for example, pUCderivatives.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants. Such plants can be grown in the normal manner and crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims.

1. An isolated, weevil-toxic protein comprising a weevil-toxic fragmentof a CryIII-class toxin obtainable from Bacillus thuringiensis strainPS50B having accession number NRRL B-21656.
 2. The protein of claim 1wherein said protein is the full-length, CryIII-class toxin obtainablefrom Bacillus thuringiensis strain PS50B having accession number NRRLB-21656.
 3. A method of controlling a weevil pest wherein said methodcomprises contacting said pest with a weevil-toxic amount of a proteinwherein said protein comprises a weevil-toxic fragment of a CryIII-classtoxin obtainable from Bacillus thuringiensis strain PS50B havingaccession number NRRL B-21656.
 4. The method according to claim 3wherein said protein is the full-length, CryIII-class toxin obtainablefrom Bacillus thuringiensis strain PS50B having accession number NRRLB-21656.
 5. The method according to claim 3 wherein said weevil is arice water weevil.
 6. A method of controlling rice water weevils whereinsaid method comprises administering to said weevils a pesticidallyeffective amount of a toxin obtainable from Bacillus thuringiensisisolate PS33F2, or a weevil-toxic fragment of said toxin.
 7. The methodaccording to claim 3 wherein said toxin, or said fragment thereof, isproduced by a plant.
 8. The method according to claim 6 wherein saidtoxin, or said fragment thereof, is produced by a plant.